c4/cam: upcoming meetings · /cam: upcoming meetings cam systems biology/ecology (new phytologist)...

C4/CAM: Upcoming Meetings

CAM Systems

Biology/Ecology (New

Phytologist) – July 15-18,

2014 Granlibakken

Conference Center- S. Lake

Tahoe, CA

Plant and Animal Genome

(PAG) Workshop –

Functional Genomics of C4

and CAM Photosynthesis,

January 11, 2014 (Saturday,

10:30 am)

Engineering CAM Photosynthetic Machinery

into Bioenergy Crops for Biofuels Production

in Marginal Environments

C4-CAM Plant Biology Meeting

University of Illinois, Urbana-Champaign

August 6-10, 2013

John C. Cushman – University of Nevada

Department of Energy, Office of Science, Genomic Science Program - Genomic Science:

Biosystems Design to Enable Next-Generation Biofuels: Award #DE-SC0008834.

Project Network

John Cushman

Karen Schlauch

James Hartwell

Xiaohan Yang

Tim Tschaplinski

Dave Weston

Gerald Tuskan

Jay Chen

Hong Guo

Anne Borland

Project Overview

Goal: Enhance WUE and adaptability

to hotter/drier climates of C3 species by

introducing CAM

Outcome: Develop new capabilities for

biomass production on marginal or

abandoned agricultural lands while

minimizing H2O and N inputs

http://www.globalwarmingart.com/

http://www.esrl.noaa.gov/

http://www.globalwarmingart.com/

http://climate.nasa.gov http://climate.nasa.gov

1960 2006

400

Rising Global Emissions, CO2, and Temperatures

Project Motivation

U.S. Drought Monitor August 14, 2012

Global Annual Groundwater Depletion

Source: United Nations Environment Program: 2012

Project Motivation

ORNL Biomass Program

http://www1.eere.energy.gov/library/default.aspx?page=1

New RFS demands feedstock enhancement/expansion

ALL soy/corn crop yield will displace ~6/12% of current

diesel/gasoline fuels

Waste stream capture would add another ~7%


0%

USDA/DOE estimates <0.3% biofuels from arid western U.S. and

0% biomass production

Geographical Distribution of Biomass Production


?

Can biomass feedstock production be expanded to more arid

areas by improving water use efficiency?

Geographical Distribution of Biomass Production

Research Question?

Can crassulacean acid metabolism

(CAM) be exploited to improve the

WUE or drought durability of (biofuel)

crops?

Existing mega-CAM species

Transfer of CAM into C3 crops

Crassulacean Acid Metabolism

C4 acid

HCO3-

Pi

CO2

Stomata Open

Stomata Closed

1° Carboxylation

Starch

PEP

PEPC

C4 acid

C3 acid

Calvin

Cycle Decarboxylation

RUBISCO DAY

NIGHT

CO2

2° Carboxylation

Physiological Consequences of CAM

Water use efficiency (WUE) is

improved 5-10-fold relative to

C4 and C3 species, respectively

The ‘CO2 pump’ of CAM

reduces photorespiration

dramatically

A more efficient RUBISCO

enzyme might improve nitrogen

use efficiency

Lüttge 2002

Robust Growth Characteristics of CAM Plants

High water use efficiency

High drought tolerance (25 mm)

Extreme heat tolerance (65°C vs. 45°C)

High light tolerance (1000 µm m-2 sec-1 PPFD)

Tolerance to UV-B irradiation

Entire shoot surface area typically

photosynthetic Borland et al. (2009) J. Exp. Bot. 60: 2879-2896.

Annual Water Demand

0

5000

10000

15000

20000

25000

30000

C3 C4 CAM

Cro

p w

ate

r d

em

an

d (

Mg

H20

ha

-1 y

ea

r-1)

Photosynthetic Pathway

Borland et al. (2009) J. Exp. Bot. 60: 2879-2896.

Above-ground Dry Biomass Productivities

Ananas comosus: ~35 Mg ha-1 year-1

Agave americana: ~42 Mg ha-1 year-1

Opuntia ficus-indica: 47-50 Mg ha-1 year-1

Nobel 1991 New Phytol. 119: 183- 205.

Annual Productivity (Above Ground)

0

10

20

30

40

50

C3C4

CAM

Ave

rag

e a

bo

ve

-gro

un

d p

rod

uctivity (

Mg

ha

-1 y

ea

r-1)

Photosynthetic Pathway

Borland et al. (2009) J. Exp. Bot. 60: 2879-2896.

Agave worldwide cultivation >500,000 ha

Large Agave species used for alcoholic beverage

production (27-38% sugar leaves/stems)

Ethanol production well developed

CAM Bioenergy Crops: Agave

Simpson et al., 2011 Global Change Biology: Bioenergy 3: 25-36. Agave americana

Agave worldwide cultivation >500,000 Ha

Large Agave species used for fiber production:

- A. sisalana (sisal) 246 x 103 Mg

- A. fourcroydes (henequin) 22 x 103 Mg

CAM Bioenergy Crops: Agave

Sisal fibers Agave sisalana

CAM Bioenergy Crops: Opuntia

Opuntia worldwide cultivation >1,000,000 ha

Large Opuntia species used for food as young

cladodes (nopalitos) and fruits (tunas) and forage

Paterson et al., (2008) Trop. Plant Biol. 1: 3-19

EPI = water index x air temperature x photosynthetic photon flux density (PPFD)

EPI predicts productivity

EPI was calculated for 87 sites in the U.S. (189 sites across the earth)

Annualized productivities of a model CAM species (Opuntia ficus-indica) were calculated

Environmental Productivity Indices (EPI) of CAM Species

Nobel (1991) Plant Cell & Environ. 14: 637-646.

Temperature Index

Water Index

PPFD Index

Temperature Index

EPI Indices Modeling of Temperature Increase

Nobel (1991) Plant Cell & Environ. 14: 637-646.

Temperature Index calculated from 10 years of daily min/max temperature data

Annualized productivities

for Opuntia ficus-indica were calculated at 2 °C, 4 °C, and 6 °C above current climate conditions

Sites with no temps below -10 °C increased by 16%, 38%, and 65%

2 °C

4 °C

6 °C

37 sites

51 sites

61 sites

CAM Species are Distributed Widely

CAM present in

~6.5% of vascular

plant species

CAM evident in more

than 35 different (10

major) plant families

Multiple independent

origins documented Silvera et al., 2010 FPB 37: 995-1010

Research Question?

Does the recurrent, independent evolution of

CAM suggest that the bar might be lower

(than C4 photosynthesis) for possible

engineering of CAM into C3 (biofuel) crops?

www.abc.net.au

Project Goals

1. Define the genetic basis of

CAM ‘modules’ in eudicot and

monocot species using

network modeling of data

derived from ‘Omics (e.g.,

transcriptomic, metabolomic)

data sets.

Opuntia ficus-indica

Agave americana

Phylogenetic Tree of Selected CAM Species

Yang Lab

Monocots

CoreEudicots

Caryophyllales

Saxifragales

Agave americana A. tequilana

A. sisalana

Kalanchoë fedtschenkoi

K. laxiflora

Opuntia ficus-indica

Mesembyranthemum crystallinum

(facultative)

Clusia minor (facultative)

Orchidaceae

Agavaceae

Phalaenopsis equestris

P. aphrodite

Cactaceae

Aizoaceae

Crassulaceae

Clusiaceae

Asparagales

Malpighiales

Sedum album (cycling)

Bromeliaceae Poales

Ananas comosus

Genomes and Transcriptomes

of Selected CAM Species

Species CAM Type

Estimated

Genome Size Transcriptome Data Reads (type) Draft Genome Data Data Source

Monocots

Agave americana Obligate 6.0 Gb 3.0 M (454), 850 M (Illumina) - X. Yang, et al., unpublished

Agave deserti Obligate 4.0 Gb 1.2 B (Illumina) - S. Gross et al., unpublished

Agave sisalana Obligate 6.4-8.5 Gb 1.5 M (454), 1.1 B (Illumina) - J. Hartwell et al., unpublished

Agave tequilana Obligate 4.0 Gb - ~30X coverage X. Yang et al., unpublished

Anana comosus Facultative 526 Mb 4.4 M (Illumina) - Ong et al., 2012

Phalaenopsis aphrodite Obligate 1.6 Gb 3.3 M (454), 290 M (Illumina) - Fu et al., 2011; Su et al., 2011

Phalaenopsis equestris Obligate 1.6 Gb 0.26 M (454), 37 M (Illumina) - Fu et al., 2011; Hsiao et al., 2011

Eudicots

Clusia minor Facultative 1.8 Gb - - A. Borland et al., unpublished

Kalanchoë fedtchenkoi Obligate 246 Mb 1.6 M (454), 900 M (SOLiD) ~60X coverage J. Hartwell et al., unpublished

Kalanchoë laxiflora Obligate 250 Mb - In progress X. Yang, et al., unpublished

Mesembryanthemum crystallinum Facultative 390 Mb 3.7 M (454), 2.4 B (Illumina) In progress J. Cushman et al., unpublished

Opuntia ficus-indica Obligate 2.8 Gb 0.6 M (454), 1.7 B (Illumina) In progress

Mallona et al., 2011; J. Cushman

et al., unpublished

Sedum album Facultative cycling 142 Mb ? (Ion Torrent™) ~46X coverage T. Michael, 2012

Agave tequilana Genome

Important obligate CAM crop

4 Gb genome (n=30) diploid

Illumina-based sequencing

completed to 30X coverage

Pac Bio-based sequencing

for assembly scaffold will be

initiated shortly

Hengfu Yin, Xiaohan Yang

Chromosome spread for

Agave. (S. Neethirajan and

J. Morrell-Falvey)

Agave sisalana Transcriptome

Important obligate CAM crop for

fiber and biofuel production

6.4 Gb genome

Roche 454 (Dark/Light) and

Illumina-based leaf development

sequencing completed

Dissection of CAM developmental

gradient along leaf axis ongoing

Phytun Bupphada, James Hartwell Poster P47

Kalanchoe laxiflora Genome

Obligate CAM model for functional

dissection of CAM

Small, short life cycle, seed producer;

transformable

146 Mb genome (n=17) diploid

Illumina-based sequencing completed

to 100X coverage; assembly at 90%

Pac Bio-based sequencing for

assembly scaffold will be initiated

shortly Hengfu Yin, Xiaohan Yang

Ice Plant Genome

Well-studied facultative CAM

halophyte model

390 Mb genome (n=9)

Illumina-based sequencing in

progress

Pac Bio-based sequencing

for assembly scaffold will be

initiated shortly

Bernie Won, Won Yim, Richard Tillett

Ice Plant Transcriptome Assembly

Hybrid assembly completed

~83% of genes

characterized (mostly from

leaf tissue)

Reference protein set Set members

(N) BLAST hit in ice plant

(%)

Eukaryotic Ultra-Conserved Orthologs 357 357 (100%)

GreenCut2 green-lineage proteins 677 675 (99%)

AVPO single-copy genes 959 947 (98%)

Arabidopsis proteome (TAIR 10) 27,416 22,740 (83%)

Won Yim, Richard Tillett Poster P49

Opuntia ficus-indica Transcriptome

Important obligate CAM crop

food and forage production

2.8 Gb genome

Illumina-based time course

sequencing in process; US vs.

water-deficit stress contrast of

mesophyll and epidermal peels.

Illumina-based C3 vs. CAM

developmental series

Jesse Mayer, Bernie Wone

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

0-2.5 2.6-5 6-10 11-15 16-20 21-25 26-30 31-35

umolH

+/gFreshW

eight

PadHeight(cm)

AcidicTitra onofOpun aficus-indica

Dawn

Dusk

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

110.00%

0 1234 56 7891011121314151617181920212223242526272829303132333435363738394041424344454647

%FreshW

eight

DaysA erWatering

rep1

rep2

rep3

Strategies for Finding CAM Genes

Developmental gradient in obligate CAM

species - “Clean System”

Hartwell Lab

1

2

3

4

5

6

CAM C3

0

50

100

150

200

250

300

350

400

450

02:00 06:00 10:00 14:00 18:00 22:00

RP

KM

Time

PPCK

C3

CAM

Strategies for Finding CAM Genes

Stress induction in facultative CAM species

– “Dirty System”

Cushman Lab CAM C3

Salt or Water Deficit Stress

TF Identification for ChIP Seq

Detailed curation and expression analysis

from microarray and Illumina data sets

followed by co-expression analysis

WTC3 WTCAM

23647 43

Bernie Won, Won Yim, Richard Tillett

Ice Plant Digital Expression Profiling

Illumina-based DEG identification of

rhythmic genes over 72 h time course (US

vs. WDS).

62

355

116

5,034 13,974 9,365

Won Yim, Richard Tillett Poster P67

TF Identification for Planned ChIP Seq

1487 putative transcription factors have been

identified.

0

20

40

60

80

100

120

140

160

180

200

AP

2/E

RF

B3 s

uperf

am

ily

BB

R-B

PC

B

ES

1

bH

LH

bZIP

C

2C

2

C2H

2

C3H

C

AM

TA

CP

P

E2F/D

P

EIL

FA

R1

GeB

P

GR

AS

G

RF

HB

H

SF

LB

D (A

S2/L

OB

) M

AD

S

MY

B s

uperf

am

ily

NA

C

NF-X

1

NF-Y

N

in-lik

e

SB

P

SR

S

TC

P

Whir

ly

WR

KY

ZF-H

D

Nu

mb

er

of

Ge

ne

s

Transcription Factor Family

Bernie Won, Won Yim, Richard Tillett Poster P67

Ice plant Transcript Mapping: Carboxylation Module

Chloroplast

Vacuole

Mitochondrion

Starch

Glycolysis

PEP MAL Malic acid

TCA Cycle

2H+

HCO3-

PEPC

CO2

CA

Stomata OPEN

Nocturnal CO2

uptake and

primary fixation

Respired CO2

MDH

H+ H+

PPi

2Pi

V-PPiase

PPCK

POH

H+ H+

ATP

ADP+Pi

V-ATPase

+ H+

Pi

OAA MAL

NADH+ NADH+

Calvin Cycle

V-MT

D D N SN SL D

0.99 **

comp23737_c1_seq21 V-ATPase V1

peripheral complex


peripheral complex

**

1.25


peripheral complex

**

0.93


peripheral complex

** *

1.19


peripheral complex

**

1.36


peripheral complex

0.19


peripheral complex

**

1.33


peripheral complex

**

1.57


peripheral complex

** **

1.00

Min Max

0.54 comp59117_c0_seq1 PEPCK

** **

D D N SN SL D

Min Max

D D N SN SL D

comp11175_c0_seq1 CA

* 2.59

2.40 comp23854_c0_seq1 PEPC

**

D D N SN SL D

D D N SN SL D

comp14442_c0_seq1 PPCK

** **

1.82

comp23866_c0_seq1 C-NAD-MDH1

* **

1.33

D D N SN SL D

comp10851_c0_seq1 AVP

-0.65 ** **

D D N SN SL D

TP

G6P

TP

Glucose

TPT

Glucose

comp2658432_c0_seq1 TPT

0.83

D D N SN SL D

comp20559_c0_seq2 PLST

1.04

D D N SN SL D

GT

G6P

GPT2

D D N SN SL D

comp11177_c0_seq1 GPT

** **

4.08

Ice plant Transcript Mapping: Dearboxylation Module

Chloroplast

Vacuole

Mitochondrion

Starch

Gluconeogenesis

PEP

MAL Malic acid

TCA Cycle

2H+

Respired CO2

ME

PPDK-RP

PYR MAL

NAD(P)H NAD(P)+

PPT

G6P

Calvin Cycle

V-TDT

Stomata CLOSED

Daytime

CO2 release

and refixation

GPT2

CO2

ATP

+ Pi

AMP

+ PPi

RUBISCO

PPDK

CO2

1.75 **

comp11165_c1_seq1 PPDK

D D N SN SL D

D D N SN SL D

comp74283_c0_seq1 TDT

** **

-1.28

comp14662_c0_seq1 PPDK-RP

* 1.40

D D N SN SL D

D D N SN SL D

comp11177_c0_seq1 GPT

** **

4.08

2.63 comp23737_c1_seq21 BASS2

** **

D D N SN SL D

** ** 0.73 comp14433_c1_seq3

NADP-ME

D D N SN SL D

Ice plant Metabolic Profiling

Chloroplast

Vacuole

Mitochondrion

TPG6P

TPG6P

Starch

Glucose

PGA

PGA

PEP

OAA

MAL MAL

Malicacid

Malicacid

OAA

MAL

FUM

PYRTCACycle

H+ H+

H+

PCRCycle

H+

ATP

ADP+Pi

Glucose

1

3

6

MAL

4

8

PYRPYR

PEP

2ATP

2ADP+Pi

CO2

RUBISCO

?

HCO3-

PEPC

CO2

PPDK

CA

NADP-ME

StomataOPENNocturnalCO2

uptakeandprimaryfixa on

StomataCLOSEDDay meCO2releaseandrefix

aon

RespiredCO2

NAD-MDH

V-ATPase

H+ H+

PPi

2Pi

V-PPiase

10

2 53

PPCKP

OH

NAD-ME

6

CO2

MAL

6

PYR

NAD-MDH

7

PYR

9


MT “CAM” MT C3 WT CAM WT C3

Organic Acids and Carbohydrates


Chloroplast

Vacuole

Mitochondrion

TPG6P

TPG6P

Starch

Glucose

PGA

PGA

PEP

OAA

MAL MAL

Malicacid

Malicacid

OAA

MAL

FUM

PYRTCACycle

H+ H+

H+

PCRCycle

H+

ATP

ADP+Pi

Glucose

1

3

6

MAL

4

8

PYRPYR

PEP

2ATP

2ADP+Pi

CO2

RUBISCO

?

HCO3-

PEPC

CO2

PPDK

CA

NADP-ME




aon

RespiredCO2

NAD-MDH

V-ATPase

H+ H+

PPi

2Pi

V-PPiase

10

2 53

PPCKP

OH

NAD-ME

6

CO2

MAL

6

PYR

NAD-MDH

7

PYR

9

malate

0 8 16 24 8 16 24 32 40 480 8 16 24 8 16 24 32 40 48

DT ZTDT ZT

0

0.5

1

1.5

2

2.5

3

oxaloacetate

0 8 16 24 8 16 24 32 40 480 8 16 24 8 16 24 32 40 48

DT ZTDT ZT

0

1

2

3

4

5

fumarate

0 8 16 24 8 16 24 32 40 480 8 16 24 8 16 24 32 40 48

DT ZTDT ZT

0

5

10

15

20

25

30

35

40

Cis-aconitate

α-ketoglutarate

Succinate

Fumarate

Citrate

Oxaloacetate

Isocitrate Malate

Pyruvate

WT US

WT DS

MUT US

MUT DS

Phosphoenol

pyruvate

pyruvate oxaloacetate

malate

fumarate succinate alpha-ketoglutarate

cis-aconitate isocitrate

citrate


Chloroplast

Vacuole

Mitochondrion

TPG6P

TPG6P

Starch

Glucose

PGA

PGA

PEP

OAA

MAL MAL

Malicacid

Malicacid

OAA

MAL

FUM

PYRTCACycle

H+ H+

H+

PCRCycle

H+

ATP

ADP+Pi

Glucose

1

3

6

MAL

4

8

PYRPYR

PEP

2ATP

2ADP+Pi

CO2

RUBISCO

?

HCO3-

PEPC

CO2

PPDK

CA

NADP-ME




aon

RespiredCO2

NAD-MDH

V-ATPase

H+ H+

PPi

2Pi

V-PPiase

10

2 53

PPCKP

OH

NAD-ME

6

CO2

MAL

6

PYR

NAD-MDH

7

PYR

9


MT “CAM” MT C3 WT CAM WT C3

Amino Acids and N-Rich Compounds

Project Goals

2. Characterize the

regulation of

‘carboxylation’,

‘decarboxylation’ and

‘stomatal control’

modules using

comparative genomics,

network/molecular

dynamics modeling,

and loss-of- function

testing.

Tissue type

Co

-exp

res

sio

n m

od

ule

ID

Dave Weston, Xioahan Yang

WGCNA Coexpression network modules for 15 different

Agave americana tissues and temporal states.

Comparative Genomics: Clustering of

Orthologous Gene Groups

Xiaohan Yang

Arabidopsis thaliana

Populus trichocarpa

Solanum tuberosum

Musa acuminata

Brachypodium distachyon

Oryza sativa

Sorghum bicolor

Zea mays

Setaria italica

Agave americana

Agave deserti

Agave tequilana

Selaginella moellendorffii

Physcomitrella patens

Chlamydomonas reinhardtii

CAM/Agave

NVP (Non-vascular

plants)

C4

C3_moncot

C3_dicot

Biological Process Enrichment in Agave

CAM

C4C3

**

Xiaohan Yang

Loss-of-function Analysis in Kalanchoë

fedtschenkoi

Rhythmic gas exchange in PPCK RNAi lines

Note loss of amplitude (amount) in PPCK RNAi

lines (green and blue); WT, Red Susie Boxall, James Hartwell Poster P69

Loss-of-function Analysis in Kalanchoë

fedtschenkoi

Reduction of PPCK activity in RNAi lines

Note loss of nocturnal kinase activity and

potentially reduced fitness in PPCK RNAi lines.

Phosphorylated PEPC PEPC

WT

PPCK1

PPCK3

2 6 10 14 18 22 2 6 10 14 18 22

Wild type PPCK RNAi

Susie Boxall, James Hartwell Poster P69 Louisa Dever….

Project Goals

3. Deploy advanced genetic

engineering technologies to

enable stacking of a large

number of transgenes to

improve transgene

persistence and to transfer

fully functional CAM ‘modules’

and ‘pathway’. In planta Gene Stacking

Henrique De Paoli, Gerald Tuskan, Xiaohan Yang

GoldenBraid: Gene Module Assembly

3a. Synthetic biology cloning system for building

genetic modules/pathways from multipartite assembly

of standard component parts:

PR (promoter), CDS (coding sequence), and TM

(terminators).

GoldenBraid Cloning System Sarrion-Perdigones et al. 2011, 2013

Enzyme A

Enzyme A

Enzyme A

Enzyme A

Enzyme A

Enzyme A Enzyme A

Enzyme A

Enzyme A Enzyme A Enzyme A Enzyme A


3b. Set of four

destination cloning

vectors (A) for

reiterative (braided)

cloning of multiple,

reusable gene

modules (B) using

the principle of

idempotency:

5 transcription unit

assemblies

demonstrated.

GoldenBraid Cloning System Sarrion-Perdigones et al. 2011, 2013


3c. Creation of

libraries of

component

elements that can

be reused.

Combinations of

different linkers and

adapters can be

used to keep the

component parts in

frame.

GoldenBraid 2.0 Cloning System Sarrion-Perdigones et al. 2013

Simplified CAM ‘Carboxylation’ Module

Mal OAA

PEP

HCO3- CO2

PEPC CA

+

NAD-MDH

PPCK

PR CDS TM EveP

R

CDS TM PR CDS TM

Eve PR CDS TM

CAM and

Succulence

Silvera et al., 2010 FPB 37: 995-1010

-30

-25

-20

-15

-10

-5

0

d13C(‰

)

PhotosynthesisType

C3

WeakCAM

StrongCAM

0

0.5

1

1.5

2

2.5

3

3.5

4

LeafThickn

ess(mm)

C3

WeakCAM

StrongCAM Survey of

173 orchid

species.

Tissue

succulence

required for

C4 acid

storage.

CAM and Succulence

Nelson and Sage, 2008

Reduced IAS is associated with (and

is likely required as an evolutionary

prerequisite) for high CAM function.

Cell size and tissue succulence are functional traits of

CAM.

CAM

C3 Reduced surface of

mesophyll exposed to

IAS (Lmes/area) limits

CO2 efflux from site of

carboxylation (Phase I

and II) and increasing

likelihood of CO2

recapture.

Can Tissue Succulence Be Engineered?

Nicolas P. et al., 2013

Can Tissue Succulence Be Engineered?

Cell size and tissue succulence in Vitis embryos are

controlled by a grape berry-specific bHLH transcription

factor that also affects expression of cell expansion genes.

Project Goals

4. To analyze the effects of

the different CAM ‘modules’

and ‘pathway’ on stomatal

control, CO2 assimilation

and transpiration rates,

WUE, and biomass yield in

Arabidopsis and Populus. Populus trichocarpa

http:rvroger.co.uk

Not represented: Section Leucoides (big leaf poplars) and Abaso (Mexican poplars).

1. Populus section Populus – aspens and White Poplar. North America, Europe, Asia.

P. tremula x alba aspen hybrid clone INRA 717 1B4 (N. Europe)

P. grandidentata – Bigtooth Aspen (N. America)

2. Populus section Aigeiros – black poplars. North America, Europe, western Asia.

P. deltoides – Eastern Cottonwood (N. America)

P. nigra – Black Poplar (Europe)

3. Populus section Tacamahaca – balsam poplars. North America, Asia.

P. maximowiczii – ‘Maximowicz' Poplar, Japanese Poplar (NE Asia)

P. trichocarpa –Black Cottonwood (Western N. America)

4. Populus section Turanga – subtropical poplars. Southwest Asia.

P. euphratica – Euphrates Poplar (SW Asia)

P. tremula x alba INRA 717 1B4

P. deltoides

P. trichocarpa

P. euphratica

P. tremula x alba INRA 717 1B4 P. tremula x alba INRA 717 1B4

P. deltoides

P. trichocarpa

P. euphratica


P. deltoides

P. trichocarpa

P. euphratica


P. deltoides

P. trichocarpa

P. euphratica

P. tremula x alba INRA717 hybrid

P. deltoides

P. trichocarpa

P. euphratica

Tuskan Lab

Leaf succulence and anatomy, cuticle

thickness, stomatal physiology, and transformability.

Candidate Populus Target Species

Project Website: CAMbiodesign.org

Home Page:

News and press releases

Publications

Events: Upcoming meetings

Position openings

Newsletter signup

Project: Research Aims

Collaborators

Data Sharing: Downloads and

supplements

Own Cloud: Internal data

sharing

KBASE: Data Analysis and Modeling

KBASE Website:

http://kbase.science.energy.org

RNA-Seq data analysis

Genome-Seq data analysis

Comparative Genomics

Coexpression networks

Regulatory network modeling

to define CAM modules

Metabolic networks

http://kbase.science.energy.org

Conclusions

Can crassulacean acid metabolism (CAM)

be engineered?

Will require a more detailed understanding of the

genetic requirements and their regulation for the

carboxylation, decarboxylation and stomatal control.

Will require a more detailed understanding of the

anatomical requirements (succulence, epidermal

waxes, stomatal density and control) of CAM.

Acknowledgements

Rebecca Albion

Travis Garcia

Jungmin Ha

Jesse Mayer

Juli Petereit

Richard Tillett

Won Yim

Bernard Wone

Rick Chen

Henrique De Paoli

Nancy Engle

Lee Gunter

Sara Jawdy

Guruprasad H. Kora

Anthony Palumbo

Zackary Moore

Mark Schuster

Heather Tran

Hengfu Yin

Susie Boxall

Phaitun Bupphada

Jack Davies

Louisa Dever

Jade Weller

Jianzhuang Yao

Project Support

Department of Energy Office of Science, Genomic Science: Biosystems Design to Enable Next-

generation Biofuels: Award #DE-C0008834

National Institutes of Health INBRE (P20 RR-016464) for Omics Centers and Bioinformatics Support

Questions?

http://CAMbiodesign.org

C4/CAM: Upcoming Meetings

CAM Systems

Biology/Ecology (New

Phytologist) – July 15-18,

2014 Granlibakken

Conference Center- S. Lake

Tahoe, CA

Plant and Animal Genome

(PAG) Workshop –

Functional Genomics of C4

and CAM Photosynthesis,

January 11, 2014 (Saturday,

10:30 am)

Project Director

John C. Cushman

Task Leaders (co-PDs)

AM Borland, J-G Chen, J Hartwell, M Martin, KA Schlauch, TJ Tschaplinski, GA Tuskan, D Weston, X Yang

External Scientific Advisory Board

Dr. James Bristow – JGI

Dr. Howard Griffiths – Cambridge University

Dr. Joe Holtum – James Cook University

Dr. Rowan Sage – University of Toronto

Dr. J.A.C. Smith – Oxford University

Dr. Stan Wullschleger – ORNL

University of Nevada, Reno (UNR)

John C. Cushman

CAM Testing in

Kalanchoë,

Arabidopsis and

Populus

GA Tuskan (ORNL)

AM Borland (ORNL)

J-G Chen (ORNL)

J Hartwell (UL)

X Yang (ORNL)

Network Modeling of CAM

Modules/KBase

DJ Weston (ORNL)

J Hartwell (UL)

AM Borland (ORNL)

J-G Chen (ORNL)

JC Cushman (UNR)

H Guo (UT), G Kora (ORNL)

KA Schlauch (UNR)

X Yang (ORNL)

CAM Metabolic &

Proteomic Profiling

TJ Tschaplinski (ORNL)

JC Cushman (UNR)

J Hartwell (UL)

M Martin (ORNL)

Metabolon Inc.

CAM Discovery

RNA/DNA/ChIP Seq

X Yang (ORNL)

JC Cushman (UNR)

J Hartwell (UL)

KA Schlauch (UNR)

University of Liverpool (UL)

James Hartwell

Oak Ridge National Laboratory (ORNL)

Xiaohan Yang

Network

Organization

Plan

INTACT System for ChIP Seq

Isolation of Nuclei Tagged in specific Cell Types

(INTACT) method allows affinity-based isolation of

nuclei from specific cell types.

K. laxiflora as target model (Bernie Wone)

A B

(Deal and Henifkoff, 2010a)

Opuntia ficus-indica Transcriptome

Comparison of well-watered and 50%

RWC loss in mesophyll and over a 24 diel

timecourse (Jesse Mayer, Bernie Wone)

Illumina-based sequencing in progress.

Metabolite study samples being submitted

this week.

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

110.00%

0 1234 56 7891011121314151617181920212223242526272829303132333435363738394041424344454647

%FreshW

eight

DaysA erWatering

rep1

rep2

rep3

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

00 10 20 30 40 50

WaterPoten

al(MPa)

%lossbyfreshweight

Project

Timeline

Aim Task Description FY13 FY14 FY15 FY16 FY17

Aim 1 Task 1.1 Transcriptome (RNA-Seq) sequencing

Task 1.2 Metabolic profiling

Task 1.3 Genomic DNA preparation, sequencing,

and genome assembly (DNA-Seq)

Task 1.4 Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq)

Aim 2 Task 2.1 Co-expression network modeling in CAM plants

Task 2.2 Gene regulatory network modeling

Task 2.3 Consolidating CAM module discovery by

computational simulation

Task 2.4 Validation of CAM module design via loss-of-function knock-down in K.

fedtschenkoi

Task 2.5 Testing of CAM module design using

transient and stable gene expression systems

Aim 3 Task 3.1 CAM gene module assembly

Task 3.2 Site-specific stacking of CAM modules in

Arabidopsis

Task 3.3 Site-specific stacking of CAM modules in

Populus

Aim 4 Task 4.1 Real-time RT-PCR expression analysis of transgenes

Task 4.2 Measurement of biochemical parameters

Task 4.3 CO2 assimilation, stomatal conductance

and transpiration rates

Task 4.4 Leaf carbon balance and level/mode of CAM activity

Task 4.5 Biomass productivity of transgenic plants

Arid Epiphytic Aquatic

Diverse Habitats of CAM Plants

Photos: Anne M. Borland

~7% of all plant species are CAM

Market Values of Economically Important CAM Species

Species Worldwide value

Pineapple $3.0 billion/yr

Vanilla (flavoring) $0.5 billion/yr

Orchids $9 billion/yr

Agave (tequila, sisal) $1.2 billion/yr

Opuntia (pads, fruits) >$100 million/yr

Horticultural and

Ornamentals >$100 million/yr

CAM Plants as Bioenergy Crops

1/3 earth’s surface area is arid or semi-arid

CAM species can be grown on marginal lands NOT suitable for C3 and C4 species (200-400 mm precipitation/year)

http://www.esa.int

40% of Global Land Area is Arid


Global Dryland Degradation


Global Annual Groundwater Depletion

Source: Gleeson et al. 2012

c4/cam: upcoming meetings · /cam: upcoming meetings cam systems biology/ecology (new phytologist)...

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